The main goal of this competitive renewal application is the full understanding of the nature of breast epithelial antigens (BEAgs) found in the circulation of breast tumors and the use of this knowledge to develop the most sensitive and specific assays for breast cancer diagnosis. Emphasis will be placed on identifying molecular and epitopic structures and maturational characteristics of BEAgs in breast cancer patient serum, so as to be able to synthesize novel recombinant epitopes, both glycosylated or not, of anti-BEAgs to participate in highly sensitive competitive recombinant epitope assays (REAs) for these breast cancer serum markers. Several of these recombinant epitopes will be expressed in fusion proteins that will participate as solid phase-incorporated antigen in REA panels. For these REAs the affinity of the participating antibody will be similar or lower for the solid phase-incorporated antigen than for competing serum breast cancer antigen glycoforms. Panels of these REAs will be created that will incorporate epitopes of BEAgs (the breast epithelial mucins, the 46 kDa Ag and conventional ones such as CEA). Sensitivity of these REAs will be heightened by protein design and molecular engineering to increase the affinity of the anti- BEAgs participating in them. This technology is represented by phage display libraries employing computer guided mutagenesis of the CDRs and by immunoglobulin chain shuffling in filamentous phage. Crucial to increasing the sensitivity of REAs is the ability acquired through this project using molecular design, to control the interplay between solid phase-incorporated fusion protein, the affinity- increased antibody and the competing breast serum antigen. The highest possibly sensitivity, while preserving the high specificity already demonstrated by the REAs, will be required to measure accurately the low levels of BEAgs to be found in the targeted subgroups potentially bearing small breast tumors usually difficult to detect: the axillary-node free patient with residual disease and the under 50 year old women, whose mammographic diagnosis is now in question. These molecularly redesigned and optimized REAs will participate as a second diagnostic test to mammograms. Their combined ability to detect small breast tumors will be determined and then compared to that of either test alone. A new area of exploration for this project will be the use of piezoelectric microsensors to increase sensitivity of detection of REAs. The advanced methodology for immunoassay optimization proposed in this project could represent a model that could have impact in the general area of serological assay development.